Approximately 20% of all married couples exhibit some form of infertility. A significant portion of the women in this group experience hypothalamic ammenohrea. For almost twenty years these women have been treated with a commercial preparation of the human gonadotropins (Pergonal.TM. of Serono), luteinizing (LH) and follicle stimulating hormone (FSH) obtained by purification of urine obtained from post-menopausal women. In general, this treatment is highly effective in stimulating folliculogenesis and steroidogenesis in the otherwise quiescent ovary. Indeed, major complications of this treatment result from the fact that this preparation works too well. The ovary can become hyperstimulated, which may result in multiple ovulations or even a life-threatening condition.
It is an aspect of the present invention to provide an improved therapeutic and treatment method which substantially reduces or eliminates such complications.
During a normal menstrual cycle, all but one of a large crop of developing follicles becomes atretic. When an artificial cycle is created by the administration of gonadotropin preparations, multiple ovulations are observed.
Although infertile couples welcome a therapy to induce the ovulatory process, they are rarely enthusiastic over the prospect of carrying 3-7 fetuses to term. An analysis of clinical, ethical and financial aspects of this complication of gonadotropic therapy is beyond the scope of this application but clearly represents issues which this invention seeks to obviate. Scientists are now beginning to understand the mechanisms responsible for the selection of the dominant follicle (the ovarian structure responsible for shedding one mature ova to be fertilized at mid cycle) which should help to reduce the occurrence of multiple pregnancies in conventionally treated woman.
The following brief summary of this area of research may prove useful in understanding the present invention.
The maturation and ultimate ovulation of only one of a large cohort of follicles is thought to be the result of continuous gonadotropin support to one follicle at the expense of all others (Richards, Rec. Prog. Hormone Res. 35:343 (1979)). Differential growth rates, blood supply, and/or secretion of stimulatory and inhibitory paracrine factors by the individual follicles have all been shown to play a role in this phenomenon. However, gonadotropin support to the ovary is an essential part of the selection process. During a normal menstrual cycle, the hypothalamus, pituitary and ovaries exist in a dynamic equilibrium. As the ovarian follicles grow and mature, they release substances that are recognized by the hypothalamus and pituitary. These hormones regulate subsequent pituitary secretions to create an endocrine environment that supports the continued growth of only one follicle. Clearly the conventional therapeutic regimen of continuous administration of exogenous gonadotropins does not replicate this finely-tuned endocrine interaction.
It is yet another aspect of the present invention to more closely simulate the natural endocrine interactions.
As stated above, gonadotropin stimulation plays a key role in follicular development and ovulation. The regulation of the absolute amount of gonadotropin secreted is one mechanism that results in the selection of a dominant follicle. The pituitary gland also adjusts the chemical composition of the gonadotropin molecules secreted during the preovulatory period. As the follicle matures, the survival time of the FSH molecule in circulation decreases. Through these mechanisms, the amount and duration of the FSH stimulus provided to the ovary is shortened. As a result, only the largest and fastest growing follicle receives continuous support.
It is now well accepted that both LH and FSH circulate as a heterogeneous population of molecules. Individual FSH isoforms exhibit identical amino acid sequences but differ in the extent to which they are post-translationally modified. As a result of these modifications, the isoforms exhibit differences in overall charge, degree of sialic acid (a terminal sugar) or sulfate incorporation, receptor binding affinity and plasma half-life (Chappel et al., Endocrine Reviews 4:179 (1983); Snyder et al. Mol. Cell. Endocrin. 54:115 (1987)).
It has been discovered that these isoforms are separable from each other on the basis of their overall charge. All of the isoforms exhibit FSH biologic activity. Isoforms that exhibit a greater net negative charge are more heavily sialylated, exhibit a longer metabolic clearance rate and a greater biologic activity due to their extended plasma survival time.
Analysis of the FSH isoforms present within commercial preparations of Pergonal has revealed the domination by the more heavily sialylated (more acidic) forms (Harlin et al., Fert. Steril. 46:1055 (1986); Chappel et al., Acta Endocrinol. 113:311 (1986)). This confirms expectations since the Pergonal preparation is derived from the urine of post-menopausal women. The pituitary gland is known to respond to low circulating levels of estradiol by producing more heavily glycosylated molecules that will survive longer in circulation (Wide and Hobson, J. Clin. Endocrinol. Metabol. 56:371 (1983)). Further, the urine would be expected to accumulate only the FSH forms that are able to survive multiple passes through the liver and kidneys and not be removed by the asialoglycoprotein receptor. The end result is that the FSH present within this commercial preparation exhibits a very prolonged serum half-life.
Although the absolute dose of exogenously administered gonadotropins can be regulated, the heterogeneity of the population within it cannot. When employed to induce follicular growth in infertile women, a number of unwanted effects may occur from the use of a preparation of long-acting FSH isoforms. The ovaries may become hyperstimulated and the patient may experience discomfort and ascites. The ovaries may hypertrophy to the point that vascular complications arise which can become life threatening. Due to the prolonged half-life of the gonadotropin, this dangerous clinical condition is difficult to manage. A second, more common complication of Pergonal administration is the occurrence of multiple ovulations. The effects of these fertility hormones upon the number of ova shed and subsequently fertilized are well known. The medical, legal and financial complications of hormone-induced multiple births are readily apparent.
To reduce the frequency of occurrence of these unwanted complications while still inducing the ovulation of a single egg, it is important to understand the reproductive endocrine events that occur during this process and attempt to mimic them. The gonadotropin stimulus provided to the ovaries during the follicular phase of the cycle changes in quantity and quality. At the onset of the cycle (at the time of menstruation) the endocrine milieu that surrounds the hypothalamo-pituitary axis is similar to a hypogonadal state. Serum gonadal steroid concentrations are low and the hypothalamo-pituitary unit responds by an increase in the quantity of FSH to be synthesized and secreted. In addition, the FSH molecule is co- and post-translationally modified such that the majority of the molecules secreted are heavily sialylated and exhibit a prolonged serum half-life. As FSH stimulates folliculogenesis and estradiol biosynthesis during the first fourteen days of the cycle, ovarian hormones (inhibin, gonadal steroids) are secreted into the peripheral circulation and feedback upon the hypothalamo-pituitary axis (Hsueh et al., Endocrine Rev. 5:76 (1984); Chappel et al., Endocrine Rev. 4:179 (1983)). The total amount of FSH to be secreted declines and the type of FSH is altered. The pituitary begins to express isoforms of FSH that exhibit a much shorter half-life, but a slightly increased receptor binding affinity.
The phenomenon of altered amount and type of FSH secreted has been discovered to be important in the process of the selection of a single dominant follicle. During the very early phase of a new ovulatory cycle, the pituitary FSH signal to the ovaries is long-lived. This acts to stimulate a large crop of primordial follicles to begin the maturational process. As this cohort of follicles begins to develop, they release hormones that signal the pituitary that the follicular growth process has begun. The pituitary responds by secreting an FSH stimulus with a shorter half-life so that only the largest (fastest growing) follicles, with the greatest number of FSH receptors, continue to receive trophic support. As this process proceeds, the largest follicles continue to grow, attract an increased blood supply and continued gonadotropin support, at the expense of the slower maturing follicles. The smaller follicles become atretic and no longer participate in the "follicular wave". After the fourteen day growth period, this process has resulted in the selection of one dominant follicle which proceeds to release one ovum to be fertilized.
When women are treated with exogenous gonadotropins prepared from the urine of post-menopausal women, they receive continuous exposure to a very long-acting isoform of FSH. Thus, the selection process to produce a single egg is not efficient, and a large number of follicles are able to survive. Since the FSH molecule produced in post-menopausal women (with low circulating levels of gonadal hormones) is predominantly the heavily sialylated form, repeated injection of long-acting FSH allow follicles to survive that ordinarily would not. Thus, the ovary can become overstimulated which may result in multiple ovulations and perhaps births. Ovarian hyperstimulation may also cause discomfort, ascites and cardiovascular complications.
It is another aspect of the present invention to provide a more physiological regimen of gonadotropin replacement to overcome the disadvantages of conventional compositions and regimens.
While more basic and less heavily sialylated forms of FSH might accomplish this, such are not readily available. Because of the presence of infectious agents in human pituitary extracts, that source of basic FSH isoforms also is unavailable. The only other source, extracts of urine obtained from post-menopausal women, contains very low proportions of the basic forms. Thus, until now it has been impossible to provide a physiologic stimulus to women with preparations heretofore available.
It is yet another aspect to provide a source of basic forms of FSH for use in more physiologically normal therapeutic regimens.